European Journal of Pharmacology - COnnecting REpositories · 2017-08-14 · Immunopharmacology and inﬂammation Reduced mucosal side-effects of acetylsalicylic acid after conjugation

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Immunopharmacology and inflammation

Reduced mucosal side-effects of acetylsalicylic acid after conjugationwith tris-hydroxymethyl-aminomethane. Synthesis and biologicalevaluation of a new anti-inflammatory compound

Gabriella Varga a, Norbert Lajkó a, Melinda Ugocsai a, Dániel Érces a, Gyöngyi Horváth b,Gábor Tóth c, Mihály Boros a,n, Miklós Ghyczy d,1

a Institute of Surgical Research, Faculty of Medicine, University of Szeged, H-6720, Szeged, Szőkefalvi-Nagy Béla u. 6, Hungaryb Department of Physiology, Faculty of Medicine, University of Szeged, H-6720, Szeged, Dóm tér 10, Hungaryc Department of Medical Chemistry, Faculty of Medicine, University of Szeged, H-6720, Szeged, Dóm tér 8, Hungaryd Pax Forschung GmbH, Im Rapsfeld 23, 50933 Cologne, Germany

a r t i c l e i n f o

Article history:Received 26 February 2016Received in revised form4 April 2016Accepted 11 April 2016

Keywords:AspirinGastritisInflammationMicrocirculationCytokinesRat

x.doi.org/10.1016/j.ejphar.2016.04.01999/& 2016 Elsevier B.V. All rights reserved.

esponding author.ail addresses: [email protected]@gmail.com (N. Lajkó), lindaugocsai@[email protected] (D. Érces),[email protected] (G. Horváth),[email protected] (G. Tóth), [email protected] (M. Ghyczy).x Forschung GmbH (Miklós Ghyczy) is then patent application EP 2889286A1 and Inte5/101501 (PCT/EP2014/078296) entitledrmacenti-inflammatory agent.

e cite this article as: Varga, G.,oxymethyl-aminomethane. Synthesis

a b s t r a c t

Acetylsalicylic acid (ASA) causes adverse haemorrhagic reactions in the upper gastrointestinal (GI) tract,and previous results have suggested that combination therapy with 2-amino-2-(hydroxymethyl)-1,3-propanediol (Tris) could provide protection in this scenario. Based on this hypothesis, our aim was todevelop a new compound from ASA and Tris precursors and to characterize the biological effects of ASA-Tris and the derivatives ASA-bis- and mono-hydroxymethyl-aminomethane (ASA-Bis, ASA-Mono, re-spectively) using in vivo and in vitro test systems.

ASA or ASA conjugates (0.55 mmol/kg, each) were administered intragastrically to Sprague-Dawleyrats. Changes in the mucosal structure and in the serosal microcirculation were detected by in vivoimaging techniques, the plasma TNF-alpha, tissue xanthine oxidoreductase and myeloperoxidase activ-ities, and liver cytochrome c changes were also determined. In two separate series, platelet aggregationand carrageenan arthritis-induced inflammatory pain were measured in control, ASA and ASA-Tris-treated groups.

Severe mucosal injury and a significant decrease in serosal red blood cell velocity developed in theASA-treated group and an �2-fold elevation in proinflammatory mediator levels evolved. ASA-Tris didnot cause bleeding, microcirculatory dysfunction, mucosal injury or an elevation in proinflammatorymarkers. The ASA-Mono and ASA-Bis conjugates did not cause macroscopic bleeding, but the in-flammatory activation was apparent. ASA-Tris did not influence the cyclooxygenase-induced plateletaggregation significantly, but the inflammatory pain was reduced as effectively as in the case of equi-molar ASA doses.

ASA-Tris conjugation is an effective approach through which the GI side-effects of ASA are controlledby decreasing the cytokine-mediated progression of pro-inflammatory events.

& 2016 Elsevier B.V. All rights reserved.

.hu (G. Varga),mail.com (M. Ugocsai),

med.u-szeged.hu (M. Boros),

Applicant and Proprietor ofrnational patent applicationutically active compound for

et al., Reduced mucosal sand biological.... Eur J Pha

1. Introduction

Aspirin or acetylsalicylic acid (ASA) is a prototype of non-ster-oidal anti-inflammatory drugs (NSAIDs), and one of the mostwidely used medications in general medical practice. Higher dosesare analgesic, antipyretic and anti-inflammatory, while lower do-ses are employed for the prevention of cardiovascular thromboticevents (Baigent et al., 2009). ASA is potent inhibitor of the con-stitutive isoform of the platelet enzyme cyclooxygenase 1 (COX-1),but it presents a variety of other pharmacological activities, suchas the reduced synthesis of coagulation factors (Meade et al., 1992;Schrör et al., 1997).

Unfortunately, the side-effects of such an efficient compound

ide-effects of acetylsalicylic acid after conjugation with tris-rmacol (2016), http://dx.doi.org/10.1016/j.ejphar.2016.04.019i

www.sciencedirect.com/science/journal/00142999

www.elsevier.com/locate/ejphar

http://dx.doi.org/10.1016/j.ejphar.2016.04.019



mailto:[email protected]












Fig. 1. Structural formulae of the developed ASA conjugates.

G. Varga et al. / European Journal of Pharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎2

are also significant. Most importantly, regular ASA usage can lead toupper gastrointestinal (GI) tract toxicity (Sørensen et al., 2000, De-laney et al., 2007). The adverse effects include mild to severe func-tional and structural damage of the mucosal layers (Appleyard et al.,2002), which can result in haemorrhage, the development of ulcersor even perforation. The causes of bleeding are usually multiple, buttwo distinct mechanisms have been revealed with some degree ofcertainty, an ASA-induced irreversible inhibition of COX-1, and anon-prostaglandin-mediated, direct topical irritation causing altera-tions in the endothelial function and mucosal permeability (Hochainet al., 2000). Moreover, ASA can influence the mitochondrial re-spiratory activity and thiol redox functions, leading to apoptosis-in-ducing signals and changes in mitochondrial membrane potential(Nulton-Persson et al., 2004; Raza et al., 2012; Redlak et al., 2005).

Many strategies for the prevention of NSAID-related ulcerativecomplications have been developed to date, such as the main-tenance of ASA in the ionic form or preventing it from dissolvinguntil it reaches the small intestine, but the efficacy of these ap-proaches is still not optimum. Hence, it is accepted that new, ef-fective formulations with reduced side-effects would be of clinicalinterest and therapeutic importance.

The amino-alcohol tris-hydroxymethyl-aminomethane (Tris),2-amino-2-(hydroxymethyl)-1,3-propanediol) has well-knownapplications in biochemistry (Gomori et al., 1955). Tris is com-monly used as a component of buffer solutions effective betweenpH 7.07 and 9.07. The Tris buffer-mediated effects are associatedwith significant influences on an arterial pH and base deficit and adecrease in the partial tension of arterial carbon dioxide, a pre-ferred alternative in patients with mixed acidosis (Hoste et al.,2005; Kallet et al., 2000). Besides an influence on acidaemia, Trismay inhibit enzyme activities, such as aminopeptidases and alpha-amylases (Desmarais et al., 2002; Ghalanbor et al., 2008).

In view of this background, our previous data and an additionalliterature review, it seemed plausible that a new drug synthesizedfrom ASA and Tris precursors might favourably affect the degree ofASA-induced mucosal damage. The primary objective of the presentstudy was to develop an agent that exhibits bioactivity against in-flammation, but is devoid of haemorrhagic side-effects in the upperGI tract. We also synthesized and tested compounds derived fromASA and mono- and bis-hydroxymethyl-aminomethane precursorsto acquire comparative information and possible clues as to themode of action. Additionally, we performed a cross-sectional eva-luation study on the analgesic and antithrombotic effectiveness ofASA-Tris as compared with ASA in the rat.

2. Materials and methods

2.1. The chemical synthesis of ASA-Mono, ASA-Bis and ASA-Trisconjugates

The synthesis of the compounds and the supporting 1H NMR

Please cite this article as: Varga, G., et al., Reduced mucosal shydroxymethyl-aminomethane. Synthesis and biological.... Eur J Pha

and HPLC studies were carried out in the Department of MedicalChemistry, University of Szeged. Details of the synthetic proce-dures and structural characterizations are described in the Sup-plementary file 1.

Briefly, ASA was dissolved in absolute tetrahydrofuran andcooled to �15 °C. Isobutyl chloroformate and triethylamine wereadded under stirring. After stirring at �15 °C for 25 min, anequimolar amount of ethanolamine, or 2-amino-1,3-propanediolor tris-hydroxymethyl-aminomethane was added and the mixturewas stirred for an additional 1 h at 0 °C, and then at room tem-perature overnight. The reaction mixtures were filtered, evapo-rated and the resulting crystalline materials were washed withdiethyl ether-hexane, resulting in the pure products (Fig. 1).

2.2. In vivo studies

The experiments were performed in three different series on atotal of 75 male Sprague-Dawley rats (average weight200 g710 g) housed in plastic cages in a thermoneutral environ-ment (2172 °C) under a 12-h dark-light cycle. The animals werekept on normal laboratory chow and then fed with a carbohy-drate-rich diet (bread rolls) for 3 days prior to the experiments.

The experimental protocols were in accordance with EU di-rective 2010/63 for the protection of animals used for scientificpurposes and were approved by the National Scientific EthicalCommittee on Animal Experimentation (National Competent Au-thority) with the licence number V./146/2013. This study alsocomplied with the criteria of the US National Institutes of HealthGuidelines for the Care and Use of Laboratory Animals.

2.2.1. Experimental protocol 135 animals were randomly allocated into 5 groups (n¼7, each).

Group 1 served as vehicle-treated control (10 ml/kg buffered0.11 M potassium hydroxide (KOH) was given orally three timesdaily on three consecutive days). In group 2, high doses of ASAsolution (0.55 mmol/kg, in a volume of 10 ml/kg; three times dailyfor 3 days) were gavaged via a flexible oesophageal tube to theanimals. After the treatments, the animals were always returnedto their cages and were fed ad libitum with a carbohydrate-richdiet.

Groups 3–5 were treated with the ASA-conjugates in equimolardoses to ASA (0.55 mmol/kg, in a volume of 10 ml/kg; three timesdaily for 3 days), group 3 was treated with ASA-Mono; group4 with ASA-Bis and group 5 with ASA-Tris.

On day 3, 2 h after the last treatments, the animals were an-aesthetized with sodium pentobarbital (50 mg/kg i.p.). For in-strumentation, the animals were placed in a supine position onheating pads, and the trachea and right jugular vein were cannu-lated to secure spontaneous breathing and i.v. administration offluids and fluorescence dye, respectively. After a midline abdom-inal incision, intravital videomicroscopy was performed to ex-amine the microcirculatory changes in the gastric serosa. The





G. Varga et al. / European Journal of Pharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎ 3

stomach was then incised along a greater curvature and rinsedwith saline to remove the gastric contents. In each group, in vivohistology of the gastric mucosa was performed by confocal laserscanning endomicroscopy (CLSEM). At the end of the protocol,tissue biopsies were obtained from the stomach and the liver, andblood samples (0.5 ml) were taken from the inferior caval vein.

2.2.2. Direct measurements on the gastric microcirculationThe orthogonal polarization spectral (OPS) imaging technique

(Cytoscan A/R, Cytometrics, Philadelphia, PA, USA) was used fornoninvasive visualization of the serosal microcirculation of thestomach. This technique utilizes reflected polarized light at thewavelength of the isobestic point of oxy- and deoxyhaemoglobin(548 nm). As polarization is preserved in reflection, only photonsscattered from a depth of 2–300 mm contribute to image forma-tion. A 10x objective was placed onto the serosal surface of thestomach, and microscopic images were recorded with an S-VHSvideo recorder 1 (Panasonic AG-TL 700; Matsushita Electric Ind.Co. Ltd, Osaka, Japan). Quantitative assessment of the micro-circulatory parameters was performed off-line by frame-to-frameanalysis of the videotaped images. Red blood cell velocity (RBCV;μm/s) changes in the postcapillary venules were determined inthree separate fields by means of a computer-assisted imageanalysis system (IVM Pictron, Budapest, Hungary).

2.2.3. In vivo detection of mucosal damageThe extent of damage of the gastric mucosa was evaluated by

means of fluorescence CLSEM (Five1, Optiscan Pty. Ltd., Mel-bourne, Victoria, Australia) developed for in vivo histology. Theanalysis was performed twice, separately by two investigators (GVand NL). The mucosal surface of the stomach was surgically ex-posed and laid flat for examination. The microvascular structurewas recorded after the iv administration of 0.3 ml of fluoresceinisothiocyanate-dextran (FITC-dextran, 150 KDa, 20 mg/ml solutiondissolved in saline, Sigma Chem.). The objective of the device wasplaced onto the mucosal surface of the stomach and confocalimaging was performed 5 min after dye administration (1 scan/image, 1024�512 pixels and 475�475 mm per image). The chan-ges in the mucosal architecture were examined following topicalapplication of the fluorescent dye acridine orange (Sigma-AldrichInc, St. Louis, MO, USA). The surplus dye was washed off the mu-cosal surface of the stomach with saline 2 min before imaging.

Non-overlapping fields gastric mucosa were processed in ASA-treated animals and compared with the samples of the control orASA conjugate-treated groups by using a semiquantitative scoringsystem as described previously (Kovács et al., 2012). We employedthree criteria: I. the structure of the microvessels (0¼normal,1¼dye extravasation, but the vessel structure recognizable,2¼destruction, and the vessel structure unrecognizable); II. oe-dema (0¼no oedema, 1¼moderate epithelial swelling, 2¼severeoedema); and III. epithelial cell outlines (0¼normal, clearly, well-defined outlines, 1¼blurred outlines, 2¼ lack of normal cellularcontours).

2.2.4. Experimental protocol 2. Platelet aggregation measurementsThe efficacy of ASA-Tris was tested on platelet functions in a

separate series; the protocol was identical to that described above.Briefly, 15 animals were randomly allocated into 3 groups (n¼5,each), group 1 served as the vehicle-treated control, and repeateddoses of ASA, or ASA-Tris solution (0.55 mmol/kg, in a volume of10 ml/kg; three times daily for 3 days) were gavaged via a flexibleoesophageal tube to the animals in groups 2 and 3, respectively.Two h after the last treatment, the animals were anaesthetizedwith sodium pentobarbital and blood samples were taken fromthe inferior caval vein. The measurement of platelet aggregationwas carried out with multiplate electrode aggregometry


(Multiplate analyzer, Roche, Basel, Switzerland). Briefly, after pla-telet activation the analyzer records the impedance between twoelectrodes reflecting thrombocyte aggregation on the surface ofthe electrodes. 300 μl blood samples were placed in hirudinizedtubes to perform multiplate aggregation tests using arachidonicacid, the substrate of COX, which subsequently forms the plateletactivator thromboxane A2 (ASPI-test), collagen, which leads to arelease of endogenous arachidonic acid and TXA2 via the collagenreceptors (Col-test), and the adenosine-diphosphate (ADP)-in-duced platelet activation test (ADP-test).

2.2.5. Experimental protocol 3The analgesic effect of ASA-Tris was tested on the carrageenan-

induced paw inflammation model, using 18 male SPRD rats ran-domly allocated into 3 groups (n¼6, each). Group 1 served aspositive control, group 2 was treated with ASA, and group 3 withASA-Tris conjugate in doses identical to those described above. Ingroups 2 and 3, the treatment was administered 3 h after the in-duction of paw inflammation by the injection of carrageenan(300 mg/30 ml) into the tibiotarsal joint of the right hind limb. Alltreatments were given to gently restrained conscious animals via a27-gauge needle without anaesthesia.

The analgesic effects of ASA and the ASA-Tris derivative treat-ments were determined by using a dynamic plantar aesthesi-ometer (mod-37450; Ugo Basile, Comerio, Italy). Before baselinemeasurements, each rat was habituated to a testing box with awiremesh grid floor for at least 20 min. The measurements wereperformed with a straight metal filament (diameter 0.5 mm) thatexerts an increasing upward force at a constant rate (4.25 g/s) witha maximum cut-off force of 50 g. The filament was placed underthe plantar surface of the right hind paw. Measurements werestopped when the paw was withdrawn, and results were ex-pressed as paw withdrawal thresholds in grams.

The baseline measurements were performed 15 min before theinduction of inflammation, while the development of inflamma-tion was investigated 3 h after the carrageenan induction. The ASAor ASA-Tris treatments were administered 10 min after the secondmeasurements; subsequent measurements were performed at 60,120 and 180 min.

2.3. In vitro studies

2.3.1. Preparation of tissue biopsiesGastric biopsies kept on ice were homogenized in phosphate

buffer (pH 7.4) containing 50 mM Tris-HCl (Reanal, Budapest,Hungary), 0.1 mM EDTA, 0.5 mM dithiotreitol, 1 mM phe-nylmethylsulfonyl fluoride, 10 μg/ml soybean trypsin inhibitor and10 μg/ml leupeptin (Sigma-Aldrich GmbH, Germany). The homo-genate was centrifuged at 4 °C for 20 min at 24,000 g and thesupernatant was loaded into centrifugal concentrator tubes(Amicon Centricon-100; 100,000 MW cut-off ultrafilter). The ac-tivity of xanthine oxidoreductase (XOR) was determined in theultrafiltered supernatant, while that of myeloperoxidase (MPO)was measured on the pellet of the homogenate.

2.3.2. Tissue MPO activityThe activity of MPO as a marker of tissue leukocyte infiltration

was measured on the pellet of the homogenate (Kuebler et al.,1996). Briefly, the pellet was resuspended in K3PO4 buffer (0.05 M;pH 6.0) containing 0.5% hexa-1,6-bis-decyltriethylammoniumbromide. After three repeated freeze-thaw procedures, the mate-rial was centrifuged at 4 °C for 20 min at 24,000 g and the super-natant was used for MPO determination. Subsequently, 0.15 ml of3,3′,5,5′-tetramethylbenzidine (dissolved in DMSO; 1.6 mM) and0.75 ml of hydrogen peroxide (dissolved in K3PO4 buffer; 0.6 mM)were added to 0.1 ml of the sample. The reaction led to the





Table 1The effects of ASA and ASA-conjugates treatment on changes of body weight [g],red blood cell velocity [mm/s] and TNF-alpha [pg/ml].

Parameters Body weight Reed blood cellvelocity

TNF-alpha

Control Median 20 963 3.0825p; 75p 12.5; 22.5 842; 1042 2.51; 3.09

ASA Median �15 269 a 6.28 a

25p; 75p �25;�7.5 224; 426 4.72; 8.57

ASA-Mono Median 15 b 539 ,a,b 4.2 a

25p; 75p 12.5; 20 502; 657 3.35; 7.82

ASA-Bis Median 15 b 432 ,a,b 1.46 ,a,b

25p; 75p 12,5; 22.5 406; 605 0.59; 2.61

ASA-Tris Median 15 b 967 b,c,d 1.94 b,c

25p; 75p 10; 20 882; 1006 0.95; 2.53

a Po0.05 between groups vs control group;b Po0.05 between ASA-Mono, ASA-Bis, ASA-Tris groups vs ASA group;c Po0.05 between ASA-Mono group vs ASA-Tris group;d Po0.05 between ASA-Bis group vs ASA-Tris.


hydrogen peroxide-dependent oxidation of tetramethylbenzidine,which could be detected spectrophotometrically at 450 nm (UV-1601 spectrophotometer; Shimadzu, Kyoto, Japan). MPO activitieswere measured at 37 °C; the reaction was stopped after 5 min bythe addition of 0.2 ml of H2SO4 (2 M) and the resulting data werereferred to the protein content.

2.3.3. XOR activityThe XOR activity was determined in the ultrafiltered, con-

centrated supernatant by a fluorometric kinetic assay based on theconversion of pterine to isoxanthopterine in the presence (totalXOR) or absence (xanthine oxidase activity) of the electron ac-ceptor methylene blue (Beckman et al., 1989).

2.3.3.1. Measurement of tissue nitric oxide (NO) products. Nitriteand nitrate (NOx), stable end-products of NO, were determined inthe gastric homogenate by the Griess reaction. This assay dependson the enzymatic reduction of nitrate to nitrite, which is thenconverted into a coloured azo compound that is detected spec-trophotometrically at 540 nm. Total NOx was calculated and ex-pressed as mmol/(mg protein) (Moshage et al., 1995).

2.3.4. Measurement of malondialdehyde (MDA) levelThe degree of lipid peroxidation was estimated via the amount

of MDA, a marker of oxidative damage of lipid membranes. MDAlevel was measured through the reaction with thiobarbituric acidand the values were corrected for the tissue protein content. TheMDA concentration was determined on a standard curve (nmol/ml) (Placer et al., 1966).

2.3.5. Measurement of cytochrome c oxidase releaseCytochrome c oxidase release was calculated via the time-de-

pendent oxidation of cytochrome c at 550 nm as described pre-viously (Szarka et al., 2004). Briefly, liver and gastric tissue sam-ples were homogenized in 10x ice-cold MitOx2 medium with aPotter grinder, and then centrifuged at 800 g for 5 min at 4 °C.50 μl supernatant was added to 2.5 ml cytochrome c stock solu-tion (10.6 mg cytochrome c dissolved in 20 ml distilled water)(Sigma-Aldrich, Budapest, Hungary) and the decrease in opticaldensity at 550 nm was measured spectrophotometrically during1-min intervals at 0, 30 and 60 min.

2.3.6. Plasma TNF-α level measurementsBlood samples (0.5 ml) were taken from the inferior caval vein

into precooled, heparizined (100 U/ml) polypropylene tubes, cen-trifuged at 1000 g at 4 °C for 30 min and then stored at �70 °Cuntil assay. Plasma TNF-alpha concentrations were determined induplicate by means of a commercially available enzyme-linkedimmunosorbent assay (Quantikine ultrasensitive ELISA kit for ratTNF-alpha; Biomedica Hungaria Kft, Budapest, Hungary). Theminimum detectable level was less than 5 pg/ml, and the inter-assay and intra-assay coefficients of variation were less than 10%.

2.4. Statistical analysis

Data analysis was performed with a statistical software package(SigmaStat for Windows, Jandel Scientific, Erkrath, Germany).Friedman repeated measures analysis of variance on ranks wasapplied within groups. Time-dependent differences from thebaseline for each group were assessed by Dunn’s method. Differ-ences between groups were analysed with Kruskal-Wallis one-way analysis of variance on ranks, followed by Dunn’s method forpairwise multiple comparison. In the Figures, median values and75th and 25th percentiles are given; P values o0.05 were con-sidered significant.


3. Results

3.1. Estimation of the severity of gastritis

ASA treatment resulted in manifest, visible bleeding and sig-nificant loss of bodyweight relative to the control group. The ASA-Mono, ASA-Bis and ASA-Tris treatments did not cause significantchanges in bodyweight (Table 1).

3.2. In vivo detection of gastric mucosal injury

The gastric microvessels were visualized by FITC-dextran ad-ministration, while the morphology of the gastric mucosa wasexamined by the topical application of acriflavine dye (Fig. 2). Inthe control group, the network of capillaries and gastric mucosalepithelium exhibited a normal pattern (M¼0; p25¼0; p75¼0.15).The evaluation of the confocal microscopic records demonstratedsignificant tissue damage in the ASA-treated group in contrastwith the control group (Fig. 3). The capillary network was severelyinjured, the fluorescent dye leakage pointed to an elevated en-dothelial permeability and oedema formation (M¼5; p25¼3.75;p75¼5.25; Figs. 2, 3). In the ASA-Mono (M¼2.5; p25¼1.8;p75¼3) and ASA-Bis (M¼2.5; p25¼2; p75¼3) treated groups,increased vascular permeability was observed, but this change wasaccompanied by a normal mucosal pattern. The ASA-Tris treat-ment did not lead to structural damage or morphological changesin the gastric mucosa. The loss of epithelium accompanying ASAadministration was not present (Figs. 2, 3), the changes were si-milar to those observed in the untreated control group (M¼0;p25¼0; p75¼0.15).

3.3. In vivo detection of the microcirculation

The RBCV of the serosa was measured as a quantitative markerof the gastric microcirculatory condition. The RBCV was sig-nificantly decreased in the ASA-treated group as compared withthe control group. ASA-Mono and ASA-Bis treatments causedsignificant, but moderate reductions in the microcirculation incontrast with the control group, and these changes were sig-nificantly lower relative to that in the ASA-treated group. The ASA-Tris treatment prevented the reduction in RBCV (Table 1).





Fig. 2. Left panel In vivo histology images of the mucosal surface of the stomachrecorded by confocal laser scanning endomicroscopy (CLSEM) after iv administra-tion of FITC-dextran: A: The mucosal vasculature in the control group. B: Dyeleakage from the vessel lumina after 3 days of ASA-treatment. C: 3 days of ASA-Mono treatment. D: 3 days of ASA-Bis treatment. E: Normal mucosal vasculatureafter 3 days of ASA-Tris-treatment. Right panel CLSEM after topical administrationof acriflavine: a: Normal structure of the mucosa in the control group. b: Total lossof epithelium on the surface after 3 days of ASA treatment. c: Normal structure ofthe mucosa after 3 days of ASA-Mono treatment. d: ASA-Bis treatment. e: Normalstructure of the mucosa after 3 days of ASA-TRIS treatment.

Fig. 3. Grading of in vivo histology. Control (white empty box), ASA-treated (greyempty box), ASA-Mono-treated (striped white box on the right side), ASA-Bis-treated (striped white box on the left side) and ASA-Tris-treated (checked whitebox) groups. The plots demonstrate the median (horizontal line in the box) and the25th (lower whisker) and 75th (upper whisker) percentiles. xPo0.05 betweengroups vs control group, #Po0.05 between ASA-treated vs ASA-Tris-treated groups.



3.4. Inflammatory enzyme activities and NOx level

The ASA treatment caused tissue leukocyte accumulation asrevealed via measurement of the MPO activity. The median MPOactivity in the control animals at the end of the observation periodwas 1035 (p25¼1020; p75¼1508) mU/(mg protein). 2 h after thelast ASA treatment, the gastric MPO activity was increased sig-nificantly (M¼2069; p25¼1951; p75¼2247 mU/(mg protein))relative to the control group. The ASA-Mono treatment resulted ina significant elevation in the MPO activity as compared with thecontrol group, while the ASA-Bis and ASA-Tris (M¼970;p25¼927; p75¼1176 mU/(mg protein)) treatments prevented theincrease of MPO activity in the gastric tissue (Fig. 4A).

The XOR enzyme activity 2 h after the last ASA treatment wassignificantly elevated in contrast with the control group. The ASA-Mono treatment did not influence while the ASA-Bis and ASA-Tristreatments significantly decreased the activity of XOR (Fig. 4B).

The ASA treatment significantly elevated the NOx level in thegastric tissue relative to the control group. The elevation of NOx

was significantly higher in comparison with the control group inthe ASA-Mono and ASA-Bis-treated groups. ASA-Tris treatmentdecreased the NOx elevation in contrast with the non-treated orother ASA conjugate-treated groups (Fig. 4C).

3.5. MDA and cytochrome c oxidase level

The MDA level was significantly increased in the gastric tissuein the ASA-treated group as compared with the control group. TheASA-Mono, ASA-Bis, or ASA-Tris treatment significantly preventedthe elevation in MDA level (Fig. 5A).

Cytochrome c release from the mitochondria as an indicator ofmitochondrial membrane damage was determined in the liver andgastric tissues. The hepatic cytochrome c level was significantlyincreased in the ASA- or ASA-Mono-treated groups as comparedwith the control group. The ASA-Bis or ASA-Tris treatment sig-nificantly prevented the elevation in cytochrome c release(Fig. 5B). The cytochrome c release was also measured in thegastric tissue (Fig. 5C). In these samples, the cytochrome c levelwas significantly elevated as a result of ASA treatment, while therelease of cytochrome c could not be demonstrated in the ASA-Trisgroup.





Fig. 4. Changes in gastric MPO activity (A), XOR activity (B) and the level of the NOx (C) in the control (white empty box), ASA-treated (grey empty box), ASA-Mono-treated(striped white box on the right side), ASA-Bis-treated (striped white box on the left side) and ASA-Tris-treated (checked white box) groups. The plots demonstrate themedian (horizontal line in the box) and the 25th (lower whisker) and 75th (upper whisker) percentiles. xPo0.05 between groups vs control group, #Po0.05 between ASA-treated vs ASA-Tris-treated groups.

Fig. 5. Malondialdehyde (MDA) level in the gastric tissues (A); cytochrome c release in the hepatic- (B) and in the gastric (C) tissues in the control (white empty box), ASA-treated (grey empty box), ASA-Mono-treated (striped white box on the right side), ASA-Bis-treated (striped white box on the left side) and ASA-Tris-treated (checked whitebox) groups. The plots demonstrate the median (horizontal line in the box) and the 25th (lower whisker) and 75th (upper whisker) percentiles. xPo0.05 between groups vscontrol group, #Po0.05 between ASA-treated vs ASA-Tris-treated groups.


3.6. Changes in plasma TNF-α level

The plasma level of TNF-α was significantly increased after ASAand ASA-Mono administration as compared with the control

Fig. 6. ASPI-test (A), ADP-test (B), Col-test results (C) in the control (white empty box), Aplots demonstrate the median (horizontal line in the box) and the 25th (lower whisker#Po0.05 between ASA-treated vs ASA-Tris-treated groups.


group. The plasma level of TNF-α in the ASA-Bis-, or ASA-Tris-treated groups was kept at a significantly lower level relative tothe ASA-treated group (Table 1).

SA-treated (grey empty box), and ASA-Tris-treated (checked white box) groups. The) and 75th (upper whisker) percentiles. xPo0.05 between groups vs control group,






3.7. Changes in platelet aggregation

To test different pathways of platelet aggregation, the ASPI-test,ADP-test and Col-test were used, which demonstrated an ap-proximately 80% decrease in platelet function after ASA treatmentas compared with the control group. After ASA-Tris treatment, theASPI-test showed that the rate of thrombocyte aggregation was20% lower than in the control group, but the Col test and ADP testresults were not affected (Fig. 6).

3.8. Changes in nociception

A significant decrease in the paw pressing force was detected inall groups 3 h after carrageenan administration on the treated side,referring to the development of inflammation-induced pain. Acute,single-dose ASA or ASA-Tris administrations significantly in-creased these values. This effect was transitional after ASA treat-ment, while equimolar ASA-Tris resulted in a sustained and pro-longed effect lasting until the end of the 180-min observationperiod (Fig. 7).

4. Discussion

Ulcerative lesions and bleeding are major side-effects of ASAtherapies in the upper GI tract. The physiological barrier me-chanism of the mucosa is vital for the homeostasis of the body(Foitzik et al., 1999), and many research attempts have thereforebeen made to reduce the incidence of haemorrhagic gastro-duodenal damage. It is well recognized that early events in thepathogenesis are microvascular leakage with leukocyte accumu-lation in the mucosa (Wallace et al., 1990). ASA might also alter thegastric mucosal cell functions (Alino et al., 1986; Pizzuto et al.,1997) and paradoxically stimulating the Ca2þ-dependent TNF-αrelease by activated macrophages which has direct cytotoxic ef-fects (Fiorucci et al., 1998).

Fig. 7. Changes in nociception in the control (white square with a thin continuous line),circle with a thin continuous line) groups. The plots demonstrate the median and the 25baseline values (0 h), xPo0.05 between groups vs control group; #Po0.05 between AS


Over recent decades, many advances have been made in thedelivery of NSAID medications to make the treatment safer. Recenttrends of drug development have included “modified release” en-teric coating formulations to prevent gastric or duodenal ulcera-tion. Co-therapy of NSAIDs with other compounds has also beenused. Is noteworthy that Tris has also been tried as a cationic salt-forming agent in 1:1 combination in the NSAID ketorolac tro-methamine, to increase the solubility of the formulation(Mroszczak et al., 1987). The main goal of the present study wasdifferent; the intention was to develop a novel anti-inflammatorycompound, with ASA-like therapeutic efficacy and reduced se-verity of GI tract-damaging side-effects. To this end, we synthe-sized a new molecule from the ASA and Tris precursors, and thedata demonstrate that the ASA-Tris conjugate works well as ananti-inflammatory compound with much less damaging effect onthe gastric mucosa as compared with the original ASA. It should beadded that the possibility of a chemical reaction occurring be-tween an NSAID and its Tris partner has never been investigatedpreviously.

In this study, we characterized the ASA-induced haemorrhagicmucosal injury via in vitro assays and in vivo tests in rodents. In-tragastric administration of ASA caused a disruption of the mu-cosal barrier, which was accompanied by deterioration of theserosal and mucosal microcirculation. The intravital histologyconfirmed the evolving endothelial injury and the associated, se-vere loss of epithelial cover. The significant role of PMN leukocytesin the deleterious effects of ASA was verified; the elevated MPOindicated that the PMNs are anchored in the inflamed gastric tis-sue. These potentially detrimental alterations were accompaniedby an enhanced activity of XOR, a major reactive oxygen species-producing enzyme. The elevated tissue MDA and NOx levels andcytochrome c release were accompanied both by increased circu-lating plasma TNF-α concentrations and by significant cytochromec release in the liver, demonstrating the spreading of inflammatorysignals and the systemic aspect of a local insult.

ASA-Tris, however, did not cause neutrophil accumulation,

ASA-treated (black triangle with a thin continuous line) and ASA-Tris treated (blackth (lower whisker) and 75th (upper whisker) percentiles.*Po0.05 within groups vsA and ASA-Tris-treated groups.






inflammatory enzyme activation or cytokine release. The ASA-Trisconjugate did not elevate the gastric MDA level, and in parallel therelease of cytochrome c could not be detected in the gastric, orhepatic tissues. The in vivo histology provided visible evidence forthe lack of mucosal damage after ASA-Tris administration. TheASA-Tris conjugate effectively blocked the increase of in-flammatory markers and weight loss, and prevented the change inmicrovascular structure too.

The treatment with the ASA-Mono compound increased theMPO and XOR activity and plasma TNF-α levels, but the body-weight of the animals did not change and the microcirculation wasonly moderately decreased. The ASA-Bis conjugate was somewhatmore effective; this treatment did not induce a mucosal injury andthe TNF-α release was lower as compared with ASA. The micro-vascular leakage could be repeatedly observed in the ASA-Monoand ASA-Bis groups, but not after the ASA-Tris treatments. Ingeneral, the RBCV changed in parallel with the damage of themicrovessels, and thus the lowest values were detected after ASAadministration; in the ASA-Mono and ASA-Bis groups, only amoderate RBCV decrease was observed, while the RBCV was keptat the control level after ASA-Tris treatment. In the ASA-Monogroup, the level of MDA was not increased, but the level of cyto-chrome c was elevated. After the ASA-Bis and ASA-Tris treatments,the MDA and the cytochrome c levels were in the normal range. Togive a better overview of the findings, the effects of ASA, ASA-Bis,ASA-Mono and ASA-Tris are summarized in a somewhat arbitrary,but more comprehensible numerical format (see Supplementaryfile 2) and these data clearly demonstrate that the reduced GItoxicity of the conjugates is in association with the specific mo-lecular structure of the substance.

The possible anti-inflammatory and antithrombotic effectswere also investigated and ASA-Tris was found to be potent inproducing an anti-nociceptive effect as ASA. The platelet responseswere assessed with validated and standardized methods (Boeeret al., 2010), and as expected, ASA treatment decreased the COX-dependent aggregation, the collagen induced aggregation and theADP-induced platelet activation. The ASA-Tris treatment decreasedthe COX-dependent aggregation to a certain degree, but it did notsignificantly influence the collagen-induced aggregation and theADP-induced platelet activation. The background of the phenom-enon demands further investigation, but this suggests that ASA-Tris is most likely not a COX inhibitor, and in this case the anti-inflammatory property stems from an effect different from ASA.

It is established that reactive oxygen species play an importantrole in the pathophysiology of acute ulceration induced by ASA(Fiorucci et al., 1998). The reactive oxygen species production canbe linked to mitochondria or to other sources, such as activatedleukocytes or the increased activity of cellular oxidases. The per-oxidation of polyunsaturated fatty acids, an immediate reactiveoxygen species-induced chain reaction, causes breakdown of thebiomembranes and the loss of maintenance of the cellular steadystate (De Cuyper and Joniau, 1980; Slater et al., 1984). The degreeof lipid peroxidation can be estimated via the amount of MDA, amarker of oxidative damage of lipid membranes. During hypoxiaor systemic inflammatory activation the progressively depressedelectron transport through the inner mitochondrial membrane isaccompanied by phospholipid damage and the loss of cytochromec. Cytochrome c, attached to the inner mitochondrial membrane,becomes detached in response to a threshold disturbance in themembrane structure, which leads to activation of the apoptoticcaspase cascade (Garrido et al., 2006).

The cytochrome c and MDA changes together are indicatorsof the reactive oxygen species-induced mitochondrial membranedamage, while NOx levels are indirectly linked to increased NOproduction. Collectively, the data demonstrate that oxidative andnitrosative stress reactions, which generally accompany


mitochondrial membrane damage, played roles in ASA-inducedmucosal injury, while ASA-Tris administration was devoid of theseconsequences. In other words, these data suggest that ASA-Tris didnot cause significant oxidative membrane damage, or the fate of thecell towards a reactive oxygen species-producing stress conditionwas blocked. Our results may also refer to a maintained or preservedenergetic state of the cell that can contribute to an improved cellfunction which finally ends in the reduction of the mucosal damage.

The mechanism of action of ASA-Tris is not known with cer-tainty. On the one hand, the masked carboxyl group in the ASAderivatives can prevent the local irritation of the gastric mucosa.While the exact mechanism of the interaction is unknown, itseems that the effect is proportional to the alcoholic moiety, sinceASA-Mono, ASA-Bis and ASA-Tris displayed an effectiveness pro-portional to the number of hydroxy groups in the compounds. Themechanism of action should be explored in further studies, but ithas already been shown that flavonoids with multiple hydroxygroups are more effective antioxidants than those with only one.Furthermore, the operation of a redox control mechanism is alsoplausible where the alcoholic moiety of ASA-Tris may be the activepart of a process which leads to a reduction of membrane damage.The reduced form of the tripeptide glutathione (GSH), and its ratioto the oxidized glutathione disulphide form (GSSG), is the majorthiol-disulphide redox buffer system of the cell. Ethanol (or per-haps analogously, ASA-Tris), through increasing the intracellularNADH can split the disulphide GSSG leading to increased GSH,thereby increasing the low GSH/GSSG back to higher levels(Watson et al., 2011). This reaction results in a swift change fromthe more oxidizing redox state towards a reducing milieu, i.e. thenormal condition of a healthy cell. Thirdly, the change in MDAsuggests a radical-induced peroxidation reaction after ASA anddirect antioxidant activity for ASA-Tris. In this case it is assumedthat in this milieu the radical, if generated, abstracts a hydrogenatom from the aromatic hydroxy group of ASA-Tris rather thanfrom a polyunsaturated fatty acid in the biomembranes of the cell.

In conclusion, we have demonstrated that a new, biologicallyactive product with promising pharmacological properties can beformed from ASA and Tris precursors. The study has a number oflimitations that may warrant discussion. For instance, the bio-chemical interactions between ASA-Tris and possible target mo-lecules need to be considered in more detail, and the reactionsassociated with reactive oxygen species generation should be in-vestigated further. Many other details remain missing at present,metabolism and tissue penetration data should be collected, butthe available evidence demonstrates that ASA-Tris might providetherapeutic benefit and efficiency by targeting inflammatorychanges without significant side-effects on the gastric mucosa.

Acknowledgements

The authors are grateful to Ms. Ágnes Fekete, Csilla Mester,Nikolett Beretka and Lilla Kovács, Zoltánné Szelepcsenyi for skilfulassistance. The study was supported by the Hungarian ScienceResearch Fund OTKA K104656. Competing interest: Pax ForschungGmbH (Miklos Ghyczy) is the Applicant and Proprietor of Eur-opean patent application EP 2889286A1 and International patentapplication WO 2015/101501 (PCT/EP2014/078296) entitled Phar-maceutically active compound for use as anti-inflammatory agent.

Appendix A. Supporting information

Supplementary data associated with this article can be found inthe online version at http://dx.doi.org/10.1016/j.ejphar.2016.04.019.


dx.doi.org/10.1016/j.ejphar.2016.04.019





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